Integrins are a family of .alpha..beta. heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al., Science 268: 233-239, 1995). Integrins also participate in signal transduction, as evidenced by either an alteration in adhesive affinity of cell surface integrins in response to cellular activation (termed inside-out signal transduction) or by affecting intracellular signaling pathways following integrin-mediated adhesion (termed outside-in signal transduction). Many biological responses are dependent at least to some extent upon integrin-mediated adhesion and cell migration, including embryonic development, hemostasis, clot retraction, mitosis, angiogenesis, cell migration, inflammation, immune response, leukocyte homing and activation, phagocytosis, bone resorption, tumor growth and metastasis, atherosclerosis, restenosis, wound healing, viral infectivity, amyloid toxicity, programmed cell death and the response of cells to mechanical stress.
The integrin family consists of 15 related known .alpha. subunits (.alpha.1, .alpha.2, .alpha.3, .alpha.4, .alpha.5, .alpha.6, .alpha.7, .alpha.8, .alpha.9, .alpha.E, .alpha.V, .alpha.IIb, .alpha.L, .alpha.M, and .alpha.X) and 8 related known .beta. subunits (.beta.1, .beta.2, .beta.3, .beta.4, .beta.5, .beta.6 .beta.7, and .beta.8). (Luscinskas et al., FASEB J. 8:929-938, 1994.) Integrin .alpha. and .beta. subunits are known to exist in a variety of pairings. Integrin ligand specificity is determined by the specific pairing of the .alpha. and .beta. subunits, although some redundancy exists as several of the integrins are known to bind the same ligand. Most integrins containing the .beta.1, .beta.2, .beta.3, .beta.5, .beta.6, and .beta.7 subunits have been found to transduce signals (reviewed by Hynes, Cell 69:11-25, 1992). Integrins are involved in both "inside-out" and "outside-in" signaling events.
Various pathologies associated with integrin-related defects are known. For example, inherited deficiencies of GP IIb-IIIa (also termed .alpha.IIb.beta.3) content or function have been described (termed Glanzmann's thrombasthenia) and are characterized by platelets that do not bind adhesive proteins and therefore fail to aggregate, resulting in a life-long bleeding diathesis. Inhibitors of the binding of fibrinogen and von Willebrand factor to GP IIb-IIIa have been described and have been found to block platelet aggregation in vitro and to inhibit clinical thrombosis in vivo (The EPIC Investigators, New England Journal of Med. 330:956-961, 1994; J. E. Tcheng et al., Circulation 91:2151-2157, 1995). Also, leukocyte adhesion deficiency (LAD) results from the absence of a .beta.2 subunit, and is characterized by leukocytes which fail to bind .beta.2 integrin ligands, resulting in individuals that are susceptible to infections.
The most studied platelet integrin .alpha.IIb.beta.3 (GPIIbIIIa) plays a critical role in homeostasis (platelet aggregation) and also in thrombosis. The .alpha.V.beta.3 plays a critical role in melanoma metastasis and angiogenesis, which is essential for cancer cell growth. The adhesion capacity of .alpha.IIb.beta.3 is known to be stimulated by various agonists such as thrombin, collagen, and ADP. This is termed inside-out signaling. There is accumulating evidence suggesting that integrins, in various cells and tissues including platelets, are also capable of mediating signals from the exterior to the cell interior, and that these signals can trigger cellular processes such as stimulating protein tyrosine phosphorylation, activating Na+/H+ antiporter, assembly of cytoskeletal structures and regulating gene expression that is involved in cell migration and proliferation. However, the mechanisms by which these signals are transmitted remain elusive. It has been hypothesized that the cytoplasmic tails of .alpha.IIb.beta.3 and other integrins may play important roles in adhesion by modulating the ligand-binding function of the extracellular domains through responses to intracellular signals generated by agonists stimulation (inside-out), and by mediating signals triggered by integrin receptor occupancy to intracellular molecules that may play a pivotal role in cellular physiological and pathological functions (outside-in).
A. Inside-Out Signaling
Inside-out signal transduction has been observed for .beta.1, .beta.2, and .beta.3 integrins. (R. O. Hynes, Cell 69:11-25, 1992; D. R. Phillips, et al. Cell 65:359-362, 1991, S. S. Smyth et al., Blood 81:2827-2843, 1993; M. H. Ginsberg, et al. Thromb. Haemostasis 70:87-93, 1993; R. L. Juliano and S. Haskill, J. Cell Biol. 120:577-585, 1993; E. Rouslahti, J. Clin. Invest. 87:1-5, 1991; Weber et al., J. Cell Biol. 134:1063-1073, 1996.)
Perhaps the most widely studied integrin that is involved in inside-out signaling is GP IIb-IIIa, the receptor for four adhesive proteins, fibrinogen, von Willebrand factor, vitronectin and fibronectin that bind to stimulated platelets (D. R. Phillips, et al., Blood 71:831-43, 1988). The binding of adhesive proteins to GP IIb-IIIa is required for platelet aggregation and normal hemostasis and is also responsible for occlusive thrombosis in high shear arteries.
GP IIb-IIIa is known to be involved in inside-out signal transduction because GP IIb-IIIa on the surface of unstimulated platelets is capable of recognizing only immobilized fibrinogen. In response to platelet stimulation by agents such as thrombin, collagen and ADP, GP IIb-IIIa becomes a receptor for the four adhesive proteins identified in the previous paragraph, and the binding of fibrinogen and von Willebrand factor causes platelets to aggregate. A monoclonal antibody has been described which detects the activated, receptor competent state of GP IIb-IIIa, suggesting that the conformation of the receptor competent form of GP IIb-IIIa differs from that of GP IIb-IIIa which does not bind soluble fibrinogen or von Willebrand factor (S. J. Shattil, et al., J. Biol. Chem. 260:11107-11114, 1985). It has been postulated that inside-out GP IIb-IIIa signal transduction is dependent on cellular proteins that act to repress or stimulate GP IIb-IIIa activation (M. H. Ginsberg, et al., Curr. Opin. Cell Biol. 4:766-771, 1992).
.beta.2 integrins on leukocytes also respond to inside-out signal transduction which accounts, for example, for the increased binding activity of LFA-1 (.alpha.L.beta..sub.2) on stimulated lymphocytes and the increased binding activity of MAC-1 (.alpha.m.beta..sub.2) on stimulated neutrophils (reviewed by T. Springer, Curr. Biol. 4:506-517, 1994).
B. Outside-In Signaling
Most integrins can be involved in outside-in signal transduction as evidenced by observations showing that binding of adhesive proteins or antibodies to integrins affects the activities of many cells, for example cellular differentiation, various markers of cell activation, gene expression, and cell proliferation (R. O. Hynes, Cell 69:11-25, 1992). The involvement of GP IIb-IIIa in outside-in signaling is apparent because the binding of unstimulated platelets to immobilized fibrinogen, a process mediated by GP IIb-IIIa, leads to platelet activation and platelet spreading (N. Kieffer and D. R. Phillips, J. Cell Biol. 113:451-461, 1991; Haimovich et al., J. Biol. Chem. 268:15868-15877, 1993).
Outside-in signaling through GP IIb-IIIa also occurs during platelet aggregation. Signaling occurs because fibrinogen or von Willebrand factor bound to the activated form of GP IIb-IIIa on the surface of stimulated platelets, coupled with the formation of platelet-platelet contacts, causes further platelet stimulation through GP IIb-IIIa signal transduction. In this manner, binding of adhesive proteins to GP IIb-IIIa can both initiate platelet stimulation or can augment stimulation induced by the other platelet agonists such as ADP, thrombin and collagen. The binding of soluble fibrinogen to GP IIb-IIIa on unstimulated platelets can also be induced by selected GP IIb-IIIa antibodies such as LIBS6 (M-M. Huang et al., J. Cell Biol. 122:473-483, 1993): although platelets with fibrinogen bound in this manner are not believed to be stimulated, such platelets will aggregate if agitated and will become stimulated following aggregation through GP IIb-IIIa signal transduction.
Outside-in integrin signal transduction results in the activation of one or more cascades within cells. For GP IIb-IIIa, effects caused by integrin ligation include enhanced actin polymerization, increased Na.sup.+ /H.sup.+ exchange, activation of phospholipases, increased phosphatidyl turnover, increased cytoplasmic Ca.sup.++, and activation of kinases. Kinases known to be activated include PKC, myosin light chain kinase, src, syk and pp125FAK. Kinase substrates identified include pleckstrin, myosin light chain, src, syk, pp125FAK, and numerous proteins yet to be identified (reviewed in E. A. Clark and J. S. Brugge, Sci. 268:233-239, 1995). Many of these signaling events, including phosphorylations, also occur in response to ligation of other integrins (reviewed in R. O. Hynes, Cell 69:11-25, 1992). Although these other integrins have distinct sequences and distinct .alpha.-.beta. parings that allow for ligand specificity, the highly conserved nature of the relatively small cytoplasmic domains, both between species and between subunits, predicts that related mechanisms will be responsible for the transduction mechanisms of many integrins.
C. Signal Transduction
The involvement of the cytoplasmic domain of GP IIb-IIIa in integrin signal transduction is inferred from mutagenesis experiments. Deletion of the cytoplasmic domain of GP IIb results in a constitutively active receptor that binds fibrinogen with an affinity equivalent to the wild-type complex, implying that the cytoplasmic tail of GP IIb has a regulatory role (T. E. O'Toole, et aL, Cell Regul. 1:883-893, 1990). Point mutations, deletions and other truncations of GP IIb-IIIa affects the ligand binding activity of GP IIb-IIIa and its signaling response (P. E. Hughes, et al., J. Biol. Chem. 270:12411-12417, 1995; J. Ylanne, et al., J. Biol. Chem. 270:9550-9557, 1995).
Chimeric, transmembrane proteins containing the cytoplasmic domain of GP IIIa, but not of GP IIb, inhibit the function of GP IIb-IIIa (Y. P. Chen et al., J. Cell Biol. 269:18307-18310, 1994), implying that free GP IIIa cytoplasmic domains bind proteins within cells which are necessary for normal GP IIb-IIa function. Several proteins have been shown to bind either the transmembrane domains or the cytoplasmic domains of GP IIb or GP IIIa.
CD-9, a member of the tetraspanin family of proteins (F. Lanza, et al., J. Biol. Chem. 266:10638-10645, 1991), has been found to interact with GP IIb-IIIa on aggregated platelets. .beta.3-endonexin, a protein identified through two hybrid screening using the cytoplasmic domain of GP IIIa as the "bait", has been found to interact directly and selectively with the cytoplasmic tail of GP IIIa (S. Shattil et al., J. Cell. Biol. 131:807-816, 1995). .beta.3-endonexin shows decreased binding to the GP IIIa cytoplasmic domain containing the thrombasthenic S752-P mutation. It is not yet known whether either of these GP IIIa-binding proteins are involved in signal transduction.
Cytoplasmic proteins that bind to .alpha.V.beta.3 have also been described which may be interacting with the integrin at the GP IIIa cytoplasmic domain sequence. Bartfeld and coworkers (N. S. Bartfeld et al., J. Biol. Chem. 268:17270-17276, 1993) used immunoprecipitation from detergent lysates to show that a MW=190-kDa protein associates with the .alpha.V.beta.3 integrin from PDGF-stimulated 3T3 cells. IRS-1 was found to bind to the .alpha.V.beta.3 integrin following insulin stimulation of Rat-1 cells stably transfected with DNA encoding the human insulin receptor (K. Vuori and E. Ruoslahti, Sci. 266:1576-1578, 1994). Kolanus et al. (Cell 86:233-242, 1996) recently identified Cytohesin-1. Cytohesin-1 specifically binds to the intracellular portion of the integrin .beta.2 chain, and overexpression of cytohesin-1 induces .beta.2 integrin-dependent binding of Jurkat cells to ICAM-1. A novel serine/threonine kinase, ILK-1, was found to associate with the .beta.1 cytoplasmic domain (Hannigan et al., Nature 379:91-96, 1996). Overexpression of ILK-1 inhibits adhesion to the integrin ligands fibronectin, laminin, and vitronectin.
Integrin binding to adhesive proteins and integrin signal transduction have a wide variety of physiological roles, as identified above. Enhanced signaling through integrins allows for increased cell adhesion and activation of intracellular signaling molecules which causes enhanced cell mobility and growth, enhanced cell responsiveness, and modulations in morphological transformations. Although integrins responsible for cellular function have been described and signaling events are beginning to be elucidated, the mechanism by which integrins transduce signals remains to be determined.
To understand the molecular mechanisms of the inside-out and outside-in signaling roles mediated by the cytoplasmic tails of .beta.3 integrin requires the identification of the intracellular molecules that interact with the intracellular tails of integrin. It has been reported that .alpha.-actinin binds to .beta.1 tails in vitro (Otey et al. J. Biol. Chem. 268:21193-21197, 1993) but the functional relevance of these bindings is not clear. By using yeast two-hybrid, ILK-1 was identified as a .beta.1 interacting protein but ILK-1 does not bind to .beta.3 (Hannigan et al., Nature 379:91-96 (1996). The present invention describes the molecular cloning of a novel human gene, Bap-1, encoding a protein, Bap-1, that associates with the integrin subunit .alpha.II and .beta.3 cytoplasmic tails. Bap-1 was also found to associate with Src kinase. The molecular isolation of Bap-1 forms the basis for the development of therapeutic agents that modulate integrin-mediated signal transduction.